The use of Fourier transform infrared spectroscopy to assay for urease from Pseudomonas aeruginosa and Canavalia ensiformis

被引:30
作者
Karmali, K
Karmali, A
Teixeira, A
Curto, MJM
机构
[1] Inst Super Engn Lisboa, Ctr Invest Engn Biotechnol, P-1949014 Lisbon, Portugal
[2] Inst Nacl Engn & Tecnol Ind, P-1649038 Lisbon, Portugal
关键词
urease from Pseudomonas aeruginosa and Canavalia ensiformis; enzyme activity; hydrolysis; urea; Fourier Transform Infrared Spectroscopy (FTIR);
D O I
10.1016/j.ab.2004.04.020
中图分类号
Q5 [生物化学];
学科分类号
071010 ; 081704 ;
摘要
A novel assay method was investigated for urease (EC 3.5.1.5) from Pseudomonas aeruginosa and Canavalia ensiformis by Fourier transform infrared spectroscopy. This enzyme catalyzed the hydrolysis of urea in phosphate buffer in deuterium oxide ((H2O)-H-2). The intensities of the bicarbonate bands maxima at 1625 and 1365 cm(-1) and of the amide I band at 1605 cm(-1) were measured as a function of time to study the kinetics of urea hydrolysis. The extinction coefficients E of urea and bicarbonate were determined to be 0.72, 0.48, and 0.56 mM(-1) cm(-1) at 1625, 1605, and 1365 cm(-1), respectively. The initial velocity is proportional to the enzyme concentration by using the ureases from both C ensiformis and P. aeruginosa. The kinetic constants (V-max, K-m, and K-cat) determined by Lineweaver-Burk plot were 532.2 U mg(-1) protein, 6.4 mM, and 806.36 s(-1), respectively. These data are in agreement with the results obtained by a spectrophotometric method using a linked assay based on glutamate dehydrogenase in aqueous media. Therefore, this spectroscopic method is highly suited to assay for urease activity and its kinetic parameters by using either cell-free extracts or purified enzyme preparations with an additional advantage of performing a real-time measurement of urease activity. (C) 2004 Elsevier Inc. All rights reserved.
引用
收藏
页码:115 / 121
页数:7
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